Many processes in the biological systems, e.g. animals and humans are mediated by the activation of proteinases. Some of these processes are constructive processes such as the blood clotting system; others are destructive, such as the food digesting enzymes in the gastrointestinal tract. Indirect evidence suggests that inappropriate or uncontrolled activation of proteolytic enzymes such as that which occurs in thrombosis or disseminated intravascular coagulation or other inflammatory conditions leads to tissue injury and human disease. The ability to identify and quantitate active proteinases in the blood and other body fluids has been limited by the fact that these enzymes when activated in blood or released by cells into fluids become complexed with various naturally occuring inhibitors.
At least eight distinct blood proteins have been characterized as inhibitors of protein cleaving enzymes. These inhibitors are also found in a variety of body fluids. The most important of these inhibitors appear to be alpha 2-plasmin inhibitor, alpha 2-macroglobulin, alpha 1-anti-trypsin, C1 inhibitor and antithrombin-heparin cofacor. These inhibitors have a broad specificity in that each of these inhibitor proteins can form a complex with a variety of proteolytic enzymes. The ability to measure these enzyme-inhibitor complexes in fluids would aid in the diagnosis and therapy of diseases involving enzyme activation. Methods to identify and to quantitate the enzyme in complex with its inhibitor have not been satisfactorily developed.
Approaches utilized by other investigators involve the production of an antibody directed against antigens in the enzyme inhibitor complex that are not shared by either one of the constituents alone. These antibodies have been produced against the alpha 2-plasmin inhibitor, plasmin complex by Plow et al. [J. Lab Clin. Med., 93:199-209 (1979)]; and against the antithrombin, thrombin complex by several investigators [Lau et al., J. Biol. Chem., 254:8751-8761 (1980)]. The former technique employs the detection of the plasmin-antiplasmin complex by latex agglutination assay, and the latter by a radioimmunoassay. These methods are promising, however, the development of an antibody in animals directed against new antigens within the enzyme-inhibitor complex has proven to be difficult.
As pointed out hereinafter alpha 2-macroglobulin (.alpha..sub.2 M), proteinase complexes are a particularly useful group of complexes within the scope of this invention. The majority of proteinases complexed to .alpha..sub.2 M loose most of their reactivity with detecting antibodies. Thus an immunologic detecting system would not permit quantitation cf the bound enzyme.
The .alpha..sub.2 M is a plasma protein with a wide spectrum of proteinase inhibiting activity [Harpel et al, Progress in Hemostasis and Thrombosis, T. H. Spaet, Ed., Grune & Stratton, Inc., New York, (1976) 3: 145-189]. It has been shown by a variety of investigators, as reviewed by Harpel, supra, and Starkey et al., Proteinases in Mannalian Cells and Tissues, A. J. Barrett, ed., Elsevier, Amsterdam, (1977) pp. 663-696, that .alpha..sub.2 M binds with a remarkably varied group of protein cleaving enzymes derived from blood, circulating white blood cells, tissues, invading bacteria, plants and snake venoms. Schultz et al., Z. Naturforsch, 10b(8):463-473 (1966), isolated .alpha..sub.2 M from human scrum and Wallenius et al., J. Biol. Chem., 225:253-267 (1957), distinguished .alpha..sub.2 M from IgM, the other serum globulin with a 19S sedimentation constant. Haverback et al., J. Clin. Invest., 41:972-980 (1962), first demonstrated the .alpha..sub.2 M bound trypsin or chymotrypsin, and observed that the hydrolytic activity of the complexed enzyme was retained against low molecular weight substrates but almost totally inhibited against large substrates such as proteins; [see also Harpel et al., J. Clin. Invest., 52:2175-2184 (1973)]. The ability of .alpha..sub.2 M to bind proteinases without completely inhibiting the active enzymatic site of its bound enzyme is distinctive as compared to other blood proteinase inhibitors that completely inactivate the active site of the proteinases.
The identification of .alpha..sub.2 M-proteinase complexes in biologic fluids has been accomplished by Ohlssen et al., Clin. Chim. Acta, 66:1-7 (1976), by his observation that the .alpha..sub.2 M enzyme complex has a different isoelectric point than does the free incomplexed .alpha..sub.2 M. Other investigators, including the inventor, have isolated .alpha..sub.2 M from blood by chromatographic techniques and have measured the activity of the putative .alpha..sub.2 M enzyme complex using small molecular weight proteinase substrates. Inherited deficiency of .alpha..sub.2 plasmin inhibitor is associated with a life-long hemorrhagic diathesis characterized by rapid dissolution of fibrin thrombi. Lijnen et al., J. Biol. Chem., 255:10214-10222 (1980) and Aoki et al., Blood, 55:483-488 (1980). In purified systems, comparing the distribution of plasmin between .alpha..sub.2 -macroglobulin, Cl inactivator and .alpha..sub.2 -antitrypsin, the .alpha..sub.2 -macroblobulin binds the majority of the enzyme; Harpel, Fed. Proc., 34:344 (1974) (Abstract). In studies from our laboratory examining the binding of plasmin in mixtures of purified .alpha..sub.2 -plasmin inhibitor and .alpha..sub.2 -macroglobulin, the plasmin inhibitor bound over 90 percent of the added plasmin; Harpel, J. Exp. Med., 146:1033-1040 (1977). These and other studies suggest that .alpha..sub.2 -plasmin inhibitor is the physiologically most important inhibitor of plasmin mediated fibrinolysis; Aoki et al., J. Clin. Invest., 60:361-369 (1977) and Aoki et al., Thrombos. Haemostas., 39:22-31 (1978).